The present invention relates to novel derivatives of avidin-type molecules, particularly to avidin-type molecules modified with 2,4,6-trinitrophenyl (TNP) or lactosyl (Lac) groups, to antigen-antibody complexes of avidin-type molecules, and to compositions comprising them for targeted diagnostic and therapeutic treatment of the liver and the reticuloendothelial system.
Drug delivery, using targeting systems wherein a drug or radioactive isotope are coupled to a specific targeting vehicle, has been studied for both therapeutic and diagnostic potentials. For the treatment of tumors, for example, targeting of drugs has been carried out via antibodies where the drug-antibody immunoconjugate is expected to localize specifically at a particular tumor cell. Limited specific uptake of immunoconjugates by human solid tumors was found to be the major limitation to drug or isotope immunotargeting, and therefore alternative vehicles for drug delivery other than antibodies showing specific affinity to a given tissue or organ, have been subject of further studies, such as, for example asialoglycoproteins for targeted delivery of small or large molecules to hepatocytes (Wu and Wu, 1993).
Streptavidin, a 52-60 kDa tetrameric non-glycosylated neutral protein which is a truncated form of the native streptavidin of Streptomyces avidinii, carries one biotin-binding site per monomer with a remarkably strong binding affinity to biotin (Chaiet and Wolf, 1964). The native, post-secretory form of streptavidin is larger and has a MW of 72 kDa (18 kDa per subunit). This native streptavidin molecule degrades rapidly to the stable 52-60 kDa streptavidin and can only be identified under special conditions. Bayer et al. (1989) reported that the native streptavidin undergoes proteolytic degradation during isolation to a truncated form with a molecular size of about 14 kDa per subunit, which is the commercial 52-60 kDa form of streptavidin known and recognized in the art. The truncation is effected through the cleavage of 12-14 amino acid residues at the N-terminal and up to 18 residues at the C-terminal end of each subunit. The 18 kDa streptavidin subunit was found to be sensitive to the action of several commercially available proteolytic enzymes, but once streptavidin (truncated form of the native molecule) is formed, it is remarkably stable to proteolytic activity (Wilchek and Bayer, 1989).
Streptavidin is similar in structure and biotin-binding properties to its counterpart avidin, a positively charged egg-white glycoprotein. The biotin-binding affinity of these two proteins is the highest recorded for any protein-ligand interaction (1015 Mxe2x88x921). Both proteins are tetramers containing one biotin-binding site per subunit and have similarity in a series of short interrupted segments (Green, 1975; Argarana et al., 1986), but they differ from each other in charge and glycosylation as well as in general primary sequence. Resistance to proteolytic enzymes is shared by both streptavidin and native avidin.
The streptavidin- and avidin-biotin complexes have provided extremely useful and versatile intermediates in a variety of biological and analytical systems (Wilchek and Bayer, 1984; Wilchek and Bayer, 1988). In principle, biotin coupled to a large variety of molecules can be recognized by avidin or streptavidin, either in their unmodified form or when coupled to various reporter probes, such as fluorescent dyes, radioactive elements, enzymes or immobilized matrices. Later, the use of these two systems has been extended to include different in vivo procedures, such as radioimmunodetection (Hnatowich et al., 1987) and immunotargeting (Longman et al., 1995). The present inventors have previously shown indirect immunotargeting of cisplatin to human epidermoid carcinoma using the avidin-biotin system (Schechter et al., 1991).
Biodistribution studies in mice comparing radioiodinated (125I)-avidin, (125I)-native streptavidin and (125I)-streptavidin showed that, at 24 h, native streptavidin had a normal clearance pattern from all organs with retention levels of 1-10% of total injected dose per gram tissue (%/g) whereas avidin was cleared at a faster rate and was in the range of 0.2-3%/g (Schechter et al., 1990). In contrast, streptavidin exhibited a remarkable and prolonged accumulation in the kidney with uptake levels of 70-80% of the injected dose/g tissue (%/g), mostly confined to the kidney cortex, for a period of 3-4 days following i.v. or i.p. injection, whereas its levels in other organs was low (0.3-4%/g) (Schechter et al., 1995). In terms of organ accumulation, 15% of the total injected dose of streptavidin was accumulated in each kidney, an organ comprising only 1% of the total body weight. Similar results of organ accumulation were also obtained for rats and rabbits (Schechter et al., 1995).
Addition of exogenous biotin did not reduce kidney uptake and did not affect streptavidin biodistribution to other organs (Schechter et al., 1990), excluding the possibility that streptavidin accumulation occurs due to interaction in the kidney with free biotin or with biotinylated proteins. The observation that native streptavidin and avidin, both displaying biotin-binding affinity, did not accumulate in the kidney, also excludes the possibility that biotin or biotinyl groups in this organ serve as the major anchor for streptavidin accumulation.
Avidins are enzyme resistant carriers (Hiller et al., 1991; Ellison et al., 1995) that can serve to provide selective and prolonged organ accumulation to ensure prolonged maintenance of these carriers in the target organ. Streptavidin itself (52-60 kDa) is accumulated in the proximal tubule of the mouse kidney for 3-4 days. This is due to processing of low MW proteins ( less than 64 kDa) which generally undergo tubular endocytosis and active lysosomal degradation. The exceptional long-term sequestration of streptavidin in the kidney is attributed to its unique resistance to enzymatic degradation (avidin, which is of a higher molecular size, 67 kDa, did not accumulate in the kidney and was rapidly cleared from the circulation and tissues).
Chemical modification of macromolecules can change the in vivo disposition profile of these macromolecules and lead to receptor-mediated targeting or other types of cellular uptake in the target organ targeted by these potential macromolecular homing devices. Hepatotropic markers which are receptor specific to terminal xcex2-D-galactose or N-acetyl-xcex1-D-galactosamine present on mammalian parenchymal cells (hepatocytes) have been reported earlier (Ashwell and Hartford, 1982; Schwartz, 1984). The high affinity interaction with this receptor triggers efficient internalization of circulating asialoglycoproteins (ASGP), synthetic glycosylated proteins, or other macromolecules (neoglycoproteins or neoglycoconjugates) modified with saccharides (Lee and Lee, 1994). Thus, carbohydrate receptor-mediated targeting to parenchymal (via terminal xcex2-D-galactose or N-acetyl-xcex1-D-galactosamine) and non-parenchymal (via terminal N-acetylglucosamine or mannose; Stahl and Schlesinger, 1989; Magnusson and Berg, 1989) cells of the liver has shown great promise as a potential delivery method using receptor-mediated endocytosis. The advantages of this system arise from the high affinity of the receptor for the ligand and the rapid recycling of the receptor molecule.
Natural asialoglycoproteins (ASGP), such as asialoorosomucoid and asialofetuin, were employed first, but later on, synthetic glycosylated proteins (neoglycoproteins) were used as prototypes of carrier systems. The rapid clearance of these carriers yielded attempts to slow down their degradation in order to achieve gradual but predominant accumulation in the target tissue. One of the approaches used chemical modification with biologically inert macromolecules, such as polyethyleneglycol (PEG). However, PEG conjugation was found to result in prolonged plasma retention due to reduction of interaction with tissues (Civitico, 1990; Crance, 1990).
Most of the systems developed for drug targeting utilize macromolecular carriers armed with targeting ligands recognized by specific cell types. Certain targeting ligands are known, the most common are terminal saccharide residues recognized by receptors on liver parenchymal (Gal and GalNac of asialoglycoproteins, Ashwell et al, 1982) non-parenchymal cells (GluNac and Man, Taylor et al, 1992), B-cells (Lasky et al, 1989) or endothelial cells (Bevilacqua et al, 1989). Recent developments in peptide chemistry and molecular biology yielded diverse peptide libraries consisting of numerous random peptide sequences (Pasqualini et al, 1996). Peptides with specific biological activity capable of mediating selective localization in tissues such as lung (Johnson et al, 1993) or lymphocytes (Cepek et al, 1994) have been obtained. An important example is the recently reported families of angiogenesis suppressing/inducing integrins that suppress or encourage the generation of new blood vessels (Varner et al, 1996; Folkman, 1996). These proteins are adhesion receptors not present in normal tissue but appear on endothelial cells of blood vessels of neovasculating areas. Since neovascularization is typical of malignant tissues at a certain stage, substances that interact with integrins might be considered as tissue markers for contrast agent delivery to blood vessels in neovaculating tumors (Brooks et al, 1994; Arap et al, 1998). Systematic screening of chemically-modified proteins (Neurath et al, 1995; Fujita et al, 1994) also yielded products recognized selectively by specific cells, for example, aromatic acid anhydrides that block CD4 cell receptors for HIV-1. Several systems were described that utilize macromolecular carriers armed with targeting ligands recognized by specific cell types (Monsigny et al, 1994; Hashida et al, 1995).
Tissue-targeting research and practice also utilize several alternative approaches. Some rely on physiochemical properties leading to passive uptake and accumulation, such as inherent accumulation of the agent by the target tissue (e.g., iodine by the thyroid). An important mechanism is the retention and enhanced permeability (EPR) phenomenon whereby molecules of a certain size may diffuse through blood vessels in areas of neovascularization as in malignant tissues (Matsumura et al, 1986; Duncan et al, 1996).
The intense activity in the field of targeting drugs to specific organs, tissues or cells (Matsumura et al, 1986) have yielded a variety of carrier systems such as pro-drugs, liposomes, e.g., sterically stabilized liposomes (SSL) (Kedar et al, 1994) or polymers, both natural and synthetic. The carrier conveys the drug to the specific tissue (via antibody or a tissue marker) where the drug executes the therapy. While tissue specific homing has been demonstrated in various studies, most macromolecular carrier systems are rapidly eliminated in the host, and consequently, these systems do not seem to exhibit a residence time necessary for achieving a therapeutic effect.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
The following abbreviations are used throughout the specification:
ASGP asialoglycoproteins
Av avidin
B biotinyl
BOC butyloxy
BSA bovine serum albumin
BT biotinyl-tyrosine
BT1 biotinyl-diaminopropyl-L-tyrosine
CDDP cis-dichlorodiamine platinum (cisplatin)
CMdex carboxymethyl dextran
DAP 1, 3-diaminopropane
DCC dicyclohexylcarbodiimide
DMF dimethylformamide
DTPA DTPAdiethylenetriaminepentaacetic acid
DDW double-distilled water
EDCI 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide
FUR 5-fluorouridine
Gd Gadolinium
Lac lactosyl
MRI magnetic resonance imaging
NHS N-hydroxysuccinimide
NL Neutralite
Ov ovalbumin
PBS phosphate-buffered saline
RES reticuloendothelial system
St streptavidin
TABAD thermophilic anhydride Brokii alcohol dehydrogenase
TNBS 2,4,6-trinitrobenzenesulfonic acid
TNP 2,4,6-trinitrophenyl
It has now been found in accordance with the present invention that a simple modification of the avidin or streptavidin molecule leads to a change in the biodistribution pattern thereof. Thus, the reaction of 2,4,6-trinitrobenzene-sulfonic acid (TNBS) with xcex5-amino groups of lysine residues in streptavidin or the lactosylation of xcex5-amino groups of lysine residues in streptavidin abolishes the accumulation of the modified streptavidin in the kidney and shifts it to the liver. The trinitrophenyl (TNP)-streptavidin product has increased liver levels as early as 1.5 h following injection, which peaks at 24-48 h to 30-50%/g tissue, and then slowly declines. The TNP-streptavidin product persists in the liver at relatively high levels for several days. Avidin, which in its native form is rapidly eliminated from all organs, also accumulates in the liver following TNP-modification, while TNP-modification of normally-cleared proteins, such as BSA, RNase, immunoglobulin (IgG) and TABAD, a relatively enzyme-resistant protein, does not result in accumulation in the liver or in any other organ at 2.5 h or 30 h. The TNP-modified avidin-type product accumulates preferentially in the Kupffer cells of the liver.
Lactosylated streptavidin is also found to accumulate in the liver at a high level that persists for several days although lactosylated avidin exhibits only short term accumulation in the liver. The lactosyl-modified avidin-type product accumulate preferentially in the liver hepatocytes.
In addition, it has also been found that when streptavidin is complexed to an anti-streptavidin antibody, the biodistribution pattern in tissues is modified and exhibits high and prolonged levels of the antigen-antibody complex in the spleen and liver, and preferentially in the cells of the reticuloendothelial system.
The present invention thus provides an avidin-type molecule, selected from native egg white avidin, recombinant avidin, deglycosylated forms of avidin, streptavidin recombinant streptavidin, and derivatives of all of the above molecules that are derivatized at sites other than the lysine and essential tyrosine residues, which avidin-type molecule is modified at the xcex5-amino groups of lysine residues with a 2,4,6-trinitrophenyl group (TNP) or with a lactosyl group, or is in a complex with an avidin-type molecule-specific antibody.
The present invention further provides radiolabeled forms of the TNP-and lactosyl-modified avidin-type molecules or of the antibody-avidin-type molecule complexes, complexes of the TNP- and lactosyl-modified avidin-type molecules or antibody-avidin-type molecule complexes of the present invention with biotinylated therapeutic, biotinylated diagnostic, biotinylated carrier-therapeutic, or biotinylated carrier-diagnostic agents, and conjugates of the TNP- or lactosyl-modified avidin-type molecules or antibody-avidin-type molecule complexes with therapeutic or diagnostic agents.
The present invention still further provides pharmaceutical compositions and methods for diagnosing and for treating hepatic disorders and disorders of the reticuloendothelial system.